Xenotransplantation (Xtr) is of interest because of the insufficient supply of human organs. The pig is considered a likely donor species for humans, but there are major barriers to successful Xtr. The first two barriers, hyperacute rejection (HAR) and acute vascular rejection (AVR), are mediated primarily by humoral immunity. HAR can be abrogated, but then AVR will occur. Strategies to interfere with AVR include reducing humoral immunity or inducing tolerance in the recipient, and decreasing immunogenicity or inducing protection in the graft. Our long-term goal is to investigate induction of protection in the donor organ against AVR. HYPOTHESIS: The cytokine IL-4 is capable of inducing broad protection against xenogeneic injury of porcine endothelial cells (ECs) and can be used to prepare an organ for Xtr, such that the organ is protected from HAR and AVR. SPECIFIC AIMS. I. Investigate protection induced by IL-4 in porcine ECs against xenogeneic activation. The activation response of ECs deficient in the main antigen responsible for graft rejection will be studied for IL-1 production and other mediators of EC activation, and the requirements and mechanisms for IL-4 protection will be defined. II. Characterize the mechanisms that participate in protection against complement killing. The participation of the signaling protein Akt is required for IL-4-induced protection. Akt is known to activate several substrates that may result in protection;therefore the identification and characterization of these pathways will be carried out. III. Investigate the role of anti-apoptotic proteins in IL-4-induced protection against apoptosis. The role of Bcl-xL and other protective molecules will be studied in protection from apoptosis elicited with TNF-alpha and with human natural killer cells. In these first three specific aims the protective pathways will be studied with kinase inhibitors and the functional role of candidate proteins will be defined with siRNA. IV. Investigate the protective effect of IL-4 overexpression against HAR and AVR in the mouse-to-rat model of Xtr. The AVR process in this animal model is similar to that in pig-to-primate models and will allow us to define the mechanisms of protection. Mouse hearts will be infected with an adenovirus carrying the IL-4 gene, or an irrelevant gene as control, and then transplanted into rats for studies of graft survival, histology, and gene expression and activation. Studies will be performed at different periods after grafting to establish the efficacy of IL-4 in preventing the manifestations of activation and the pathological course of rejection. It is expected that there will be an attenuation of the intensity and a delay in the manifestation of such processes, and prolongation of graft survival. Accomplishing these goals will result in understanding the mechanisms of protection of IL-4 in donor organs from a different species and to what extent they contribute to successful Xtr, which is essential for those patients with end-stage organ failure for whom no human organs are available. PUBLIC HEALTH RELEVANCE The supply of human organs for transplantation is insufficient, and a potential solution to overcome this shortage is xenotransplantation (using organs from donor animals such as the pig). Accomplishing the goals of this proposal will define a novel approach to modify donor organs for clinical xenotransplantation. This will also provide knowledge for new treatments for vascular diseases, including ischemia and atherosclerosis.